Chimeric isoprenoid synthases and uses thereof

ABSTRACT

Disclosed is a chimeric isoprenoid synthase polypeptide including a first domain from a first isoprenoid synthase joined to a second domain from a second, heterologous isoprenoid synthase, whereby the chimeric isoprenoid synthase is capable of catalyzing the production of isoprenoid reaction products that are not produced in the absence of the second domain of the second, heterologous isoprenoid synthase. Also disclosed is a chimeric isoprenoid synthase polypeptide including an asymmetrically positioned homologous domain, whereby the chimeric isoprenoid synthase is capable of catalyzing the production of isoprenoid reaction products that are not produced when the domain is positioned at its naturally-occurring site in the isoprenoid synthase polypeptide.

This application is a continuation of U.S. application Ser. No.09/576,057 filed May 23, 2000, now abandoned which is a continuation ofU.S. application Ser. No. 09/514,513, filed Feb. 28, 2000, which is adivisional of U.S. application Ser. No. 09/134,699, filed Aug. 14, 1998,now U.S. Pat No. 6,072,045 which is a continuation of U.S. applicationSer. No. 08/631,341, filed Apr. 12, 1996 (now U.S. Pat. No. 5,824,774).

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made in part with Government funding, and theGovernment therefore has certain rights in the invention.

BACKGROUND OF THE INVENTION

This invention relates to modified isoprenoid synthase enzymes, theirencoding genes, and uses thereof.

The term isoprenoid is used to refer to a family of compounds derivedfrom the isoprene building block. In particular, plant isoprenoidscomprise a structurally diverse group of compounds that can be dividedinto classes of primary and secondary metabolites (FIG. 1). Isoprenoidsthat are primary metabolites include sterols, carotenoids, growthregulators, and the polyprenol substituents of dolichols, quinones, andproteins. These compounds are essential for membrane integrity,photoprotection, orchestration of developmental programs, and anchoringessential biochemical functions to specific membrane systems,respectively. Isoprenoids that are classified as secondary metabolitesinclude monoterpenes, sesquiterpenes, and diterpenes. These compoundsare said to mediate important interactions between plants and theirenvironment. For example, specific terpenoids have been correlated withplant-plant (Stevens, In: Isopentoids in Plants, Nes, W. D. Fuller, G.,and Tsai, L.-S., eds., Marcel Dekker, New York, pp. 65-80, 1984),plant-insect (Gibson and Pickett, Nature 302:608, 1983), andplant-pathogen interactions (Stoessl et al., Phytochemistry 15:855,1976).

The common denominator for this diverse array of compounds is theiruniversal five-carbon building block, isoprene. The “biogenic isoprenerule” was employed to rationalize the biosynthetic origins of allterpenoids derived from isoprene (Ruzicka, Experientia 10:357, 1953).The polymerization of two diphosphorylated isoprene building blocks(e.g., IPP and dimethylallyl) generates geranyl diphosphate (GPP), alinear C10 intermediate that can be converted to cyclic or linearend-products representing the monoterpenes, or used in another round ofpolymerization. The addition of a third isoprene unit to GPP generatesfarnesyl diphosphate (FPP), which can also be converted to cyclic orlinear products representing the sesquiterpene class. Continuing thepolymerization and chemical differentiation cycle leads to theproduction of other classes of terpenoids named according to the numberof isoprene building blocks leading to their biosynthesis, for example,the addition of a third IPP to FPP generates geranylgeranyl diphosphate(GGPP).

These polymerization reactions are catalyzed by prenyltransferases thatdirect the attack of a carbocation (an electron deficient carbon atomresulting from the loss of the diphosphate moiety of one substrate) toan electron-rich carbon atom of the double bond on the IPP molecule(FIG. 2). The electrophilic nature of these reactions is said to beunusual relative to more general nucleophilic condensation reactions,but this appears to be a common reaction among isoprenoid biosyntheticenzymes and especially those enzymes involved in catalyzing thecyclization of various isoprenoid intermediates (Gershenzon and Croteau,In: Lipid Metabolism in Plants, Moore, T. S., ed., CRC Press, BocaRaton, Fla., pp. 340-388). The enzymes responsible for the cyclizationof GPP, FPP, and GGPP are referred to as monoterpene, sesquiterpene, andditerpene synthases or synthases, and represent reactions committingcarbon from the general isoprenoid pathway to end products in themonoterpene, sesquiterpene, and diterpene classes, respectively.

Two important biochemical distinctions between the prenyltransferase andsynthase reactions are illustrated in FIG. 2. The prenyltransferasescatalyze carbon-carbon bond formation between two substrate molecules,whereas the synthases catalyze an intramolecular carbon-carbon bondformation. The prenyltransferases also catalyze reactions with verylittle variance in the stereochemistry or length of the ensuing polymer.Prenyltransferases differ in the length of the allyic substrates thatcan be accepted in initiating these reactions. The synthases are alsosubstrate specific. However, diverse sesquiterpene synthases, forexample, can utilize the same substrate to produce different reactionproducts.

The biosynthesis of isoprenoids such as cyclic terpenes is said to bedetermined by key branch point enzymes referred to as terpene synthases.The reactions catalyzed by terpene synthases are complex, intramolecularcyclizations that may involve several partial reactions. For example,the bioorganic rationale for the cyclization of FPP by two sesquiterpenesynthases are shown in FIG. 3. In step 1, the initial ionization of FPPis followed by an intramolecular electrophillic attack between thecarbon bearing the diphosphate moiety and the distal double bond to formgermacene A, a macrocylic intermediate. Internal ring closure andformation of the eudesmane carbonium ion constitutes step 2. For tobacco5-epi-aristolochene synthase (TEAS), the terminal step is a hydrideshift, methyl migration, and deprotonation at C9 giving rise to5-epi-aristolochene as depicted in step 3a. Hyoscyamus muticusvetispiradiene synthase (HVS) shares a common mechanism at steps 1 and2, but differs from TEAS in the third partial reaction in which a ringcontraction would occur due to alternative migration of an electronpair. In each case, a monomeric protein of approximately 64 kD catalyzesthe complete set of partial reactions and requires no cofactors otherthan Mg⁺².

SUMMARY OF THE INVENTION

In general, the invention features a chimeric isoprenoid synthasepolypeptide including a first domain from a first isoprenoid synthasejoined to a second domain from a second, heterologous isoprenoidsynthase, whereby the chimeric isoprenoid synthase is capable ofcatalyzing the production of isoprenoid reaction products that are notproduced in the absence of the second domain of the second, heterologousisoprenoid synthase. In preferred embodiments, the chimeric isoprenoidsynthase is capable of catalyzing at least two different isoprenoidreaction products; the isoprenoid reaction products are cyclic; thesecond domain of the second, heterologous isoprenoid synthase alsodetermines the ratio of the isoprenoid reaction products of the chimericisoprenoid synthase; the first domain from the first isoprenoid synthaseis a plant isoprenoid synthase and the second domain from the second,heterologous isoprenoid synthase is also from a plant isoprenoidsynthase.

Preferably, the chimeric isoprenoid synthase is chosen from the groupconsisting of (a) the tobacco-Hyoscyamus CH4 chimeric isoprenoidsynthase; (b) the tobacco-Hyoscyamus CH10 chimeric isoprenoid synthase;(c) the tobacco-Hyoscyamus CH11 chimeric isoprenoid synthase; (d) thetobacco-Hyoscyamus CH12 chimeric isoprenoid synthase; (e) thetobacco-Hyoscyamus CH13 chimeric isoprenoid synthase; or (f) thetobacco-Hyoscyamus CH14 chimeric isoprenoid synthase, all as describedherein.

In preferred embodiments, the chimeric isoprenoid synthase catalyzes theproduction of an isoprenoid reaction product that is of agricultural,pharmaceutical, commercial, or industrial significance (e.g., anantifungal agent, antibacterial agent, or antitumor agent).

In other related aspects, the invention features DNA, vectors, and cells(for example, E. coli, Saccharomyces cerevisiae, animal or plant cells)encoding or containing a chimeric isoprenoid synthase polypeptide.

In another aspect, the invention features a chimeric isoprenoid synthasepolypeptide including an asymmetrically positioned homologous domainwhereby the chimeric isoprenoid synthase is capable of catalyzing theproduction of isoprenoid reaction products (preferably, cyclic products)when the domain is positioned at its naturally-occurring site in theisoprenoid synthase polypeptide.

In another aspect, the invention features a method for producing achimeric isoprenoid synthase polypeptide, the method involving: (a)providing a cell transformed with DNA encoding a chimeric isoprenoidsynthase positioned for expression in the cell; (b) culturing thetransformed cell under conditions for expressing the DNA; and (c)recovering the chimeric isoprenoid synthase.

By “isoprenoid synthase” is meant a polypeptide that is capable ofcatalyzing a reaction involving the intramolecular carbon-carbon bondformation of an allylic diphosphate substrate (for example, a C₁₀, C₁₅,or C₂₀ allylic diphosphate substrate) to an isoprenoid product (forexample, a monoterpene, diterpene, sesquiterpene, or sterol product).Examples of such isoprenoid synthases include, without limitation,monoterpene synthases (for example, limonene synthase), diterpenesynthases (for example, casbene synthase), and sesquiterpene synthases(for example, 5-epi-aristolochene synthase, vetispiradiene synthase, andcadinene synthase) that are responsible for cyclization of geranyldiphosphate (GPP), farnesyl diphosphate (FPP), and geranylgeranyldiphosphate (GGPP), respectively. A number of terpene synthases fromplant and microbial sources have been isolated and characterized (see,for example, Moestra and West, Arch. Biochem. Biophys. 238:325, 1985;Hohn and Van Middlesworth, Arch. Biochem. Biophys. 251:756, 1986; Hohnand Plattner, Arch. Biochem. Biophys. 272:137, 1989; Cane and Pargellis,Arch. Biochem. Biophys. 254:421, 1987; Munck and Croteau, Arch. Biochem.Biophys. 282:58, 1990; Alonso et al., J. Biol. Chem. 267:7582, 1992;Savage et al., J. Biol. Chem. 269:4012, 1994; Croteau et al., Arch.Biochem. Biophys. 309:184, 1994; Vögeli et al., Plant Physiol. 93:182,1990; Guo et al., Arch. Biochem. Biophys. 308:103, 1994; and Gamblieland Croteau, J Biol. Chem. 259:740, 1984). In general, terpene synthasesare soluble enzymes having a molecular weight of about 40 to 100 kD.Genes encoding a number of monoterpene, diterpene, and sesquiterpenesynthases have been described for a number of plant and microbialorganisms (see, for example, Hohn and Beremand, Gene 79:131, 1989;Proctor and Hohn, J Biol. Chem. 268:4543, 1993; Facchini and Chappell,Proc. Natl. Acad. Sci. 89:11088, 1992; Back and Chappell, J Biol. Chem.270:7375, 1995; Colby et al., J. Biol. Chem. 268:23016, 1993; Mau andWest, Proc. Natl. Acad. Sci. 91:8497, 1994; Chen et al., Arch. Biochein.Biophys. 324:255, 1994; and Cane et al., Biochemistry 33:5846, 1994).

By “polypeptide” or “protein” is meant any chain of amino acids,regardless of length or post-translational modification (for example,glycosylation or phosphorylation).

By “joined to” is meant covalently bonded either directly or indirectly(i.e., the domains are separated by an intervening amino acid sequence).Such domains may be bonded by any means, including, without limitation,a peptide bond or chemical linkage.

By “domain” is meant a contiguous stretch of amino acids within apolypeptide or protein.

By “isoprenoid” is meant a compound that is derived from an isoprenebuilding block. In particular, isoprenoid compounds include, withoutlimitation, monoterpenes, diterpenes, sesquiterpenes, and sterols. Asdescribed herein, isoprenoids are found in a variety of organisms, forexample, animal, fungal, or bacterial sources.

By “asymmetrically positioned” is meant located within the chimericpolypeptide at a site which differs from its position in thenaturally-occurring polypeptide.

By “heterologous” is meant derived from different sources (in this case,different polypeptides).

By “homologous” is meant derived from the same source (in this case, thesame polypeptide).

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims.

DETAILED DESCRIPTION

The drawings will first be described.

DRAWINGS

FIG. 1 is a schematic illustration showing the isoprenoid biosyntheticpathway with respect to the type of end products and their respectivephysiological functions. Broken arrows indicate multiple steps orreactions.

FIG. 2 is a schematic illustration showing the various reactions thatare catalyzed by prenyltransferases and terpene synthases.

FIG. 3 is a schematic illustration showing a reaction mechanism for thesynthesis of eremophilane (tobacco 5-epi-aristolochene synthase, TEAS)and vetispiradiene (Hyoscyamus vetispiradiene synthase, HVS) typesesquiterpene synthases. Partial reactions 1 and 2 are considered commonto both types of synthases. Mechanistic differences in partial reactions3a and 3b are sufficient to account for the different reaction productsshown.

FIG. 4A is a schematic illustration showing the chimeric constructs usedto map catalytic domains within sesquiterpene synthases. Line drawingsdepict composite diagrams for wildtype (i.e., TEAS and HVS) and chimeric(CH1-CH14) sesquiterpene synthase genes that were engineered into thebacterial expression vector pGBT-T19. Gene constructs were preparedusing a combination of the available restriction endonuclease sites andamplification of select regions using PCR and PCR primers harboringconvenient restriction endonuclease sites. Correspondence between uniquerestriction endonuclease sites and amino acid positions are noted.

FIG. 4B is a photograph of a TLC experiment showing synthase enzymeactivities in sonicated lysates of E. coli TB1 cells expressing theTEAS, HVS, and chimeric synthase constructs (CH1-CH14) and measuredusing ³H-FPP. Reaction products were separated by argentation-TLC anddetected by autoradiography. The radioactivity in 0.5 mm segments ofeach lane of an argentation-TLC plate was determined in a scintillationcounter, and radioactivity associated with the zones for the TEAS andHVS specific products was set to 100%.

FIG. 5 is a schematic illustration showing the correspondence betweenexons and functional domains within isoprenoid synthases. The upperdiagram represents the organization of exons within the TEAS gene, whichis nearly identical to that of the HVS and casbene synthase genes. Thelower diagram shows the alignment of functional domains to the exonicorganization of the TEAS and HVS genes. Exon numbers are shown withinthe upper diagram, and all other numbers refer to amino acid positions,some of which correspond to the noted restriction endonuclease sites.

FIG. 6 is a schematic diagram showing a domain switching strategy usedto generate a quiescent synthase (QH1). Substituting the inactive HVSdomain corresponding to exon 4 into CH3 results in a synthase having analtered enzyme activity.

FIG. 7 is a schematic diagram of a domain switching strategy used forproducing a chimeric quiescent-casbene synthase, and possible reactionproducts.

FIG. 8 is a schematic illustration of a domain switching strategy forproducing a chimeric quiescent-cadinene synthase, and possible reactionproducts.

Chimeric Isoprenoid Synthases

Plasmids designed for expressing a chimeric synthase were generated bysubstituting a portion of a gene encoding a domain from tobacco5-epi-aristolochene synthase with a portion of a gene encoding a domainfrom Hyoscyamus vetispiradiene synthase. These plasmids were expressedin bacteria, and bacterial lysates were prepared and assayed forsesquiterpene synthase activity. The sesquiterpene synthase assaysincluded an argentation-thin layer chromatography (TLC) analysis whichdistinguished the aristocholene and vetispiradiene reaction products(Back and Chappell, J. Biol. Chem. 270:7375, 1995). As shown in FIG. 4A,fourteen chimeric synthase constructs were generated and were assayed asfollows.

Full-length cDNAs for the tobacco 5-epi-aristolochene synthase (TEAS)and Hyoscyamus vetispiradiene synthase (HVS) were cloned into theEcoRI/XhoI sites of pBluescript SK (Stratagene), creating the pBSK-TEASand pBSK-HVS plasmids, respectively (Back and Chappell, J Biol. Chem.270:7375, 1995). The TEAS and HVS cDNA inserts of these expressionplasmids were oriented with their translation start codons neighboringthe EcoRI restriction site and their 3′ poly A tail flanked by the XhoIrestriction site of the pSK plasmid.

Chimeric synthases CH1, CH2, CH5, and CH7 were constructed by utilizingthe conserved HindIII and NdeI restriction sites found between thetobacco and Hyoscyamus genes. CH1 was prepared by ligating the 5′terminal portion of the TEAS gene (corresponding to the EcoRI to HindIIIfragment) with the 3′ terminal portion of HVS gene (corresponding to theHindIII to KpnI fragment) into the bacterial expression vector pGBT-T19(Gold Biotechnology) predigested with EcoRI and KpnI.

CH2 was prepared by ligating the 5′ terminal portion of the TEAS gene(corresponding to the EcoRI to NdeI fragment) with the 3′ terminalportion of HVS gene (corresponding to the NdeI to KpnI fragment) intopGBT-T19.

CH5 was prepared by ligating the 5′ terminal portion of the HVS gene(corresponding to the EcoRI to HindIII fragment) with the 3′ terminalportion of TEAS gene (corresponding to the HindIII to KpnI fragment)into pGBT-T19.

CH7 was prepared by ligating the 5′ terminal portion of the HVS gene(corresponding to the EcoRI to NdeI fragment) with the 3′ terminalportion of TEAS gene (corresponding to the NdeI to KpnI fragment) intopGBT-T19.

CH3, CH4, CH12, and CH13 were constructed using conventional polymerasechain reaction (PCR) methodologies, with primers designed for theamplification of particular segments of the HVS gene. To facilitatedirectional cloning and maintenance of reading frame, primers were alsodesigned to contain convenient restriction sites.

CH3 was constructed as follows. An EcoRI/ClaI restriction fragment ofthe TEAS gene was isolated and ligated to the ClaI/KpnI fragment of theHVS gene. The HVS ClaI/KpnI fragment was prepared by PCR methodologyusing5′-d(GGGATCGATGACATAGCCACGTATGAGGTT)-3′ (SEQ ID NO: 1; ClaIrestriction site underlined) as the forward primer and 5′-d(AATACGACTCACTATAG)-3′ (SEQ ID NO: 2) as the reverse primer(corresponding to the T7 sequence found in the multiple cloning site ofpBSK) using pBSK-HVS as the DNA template. The resulting restrictionfragment was ligated into the EcoRI/KpnI sites of the pGBT-T19 vector.

CH4 and CH13 were constructed in a similar manner, but using the forwardamplification primers 5′-d(CGAGTCAACATGGTTTATTGAGGGATA)-3′ (SEQ ID NO:3; HincII restriction site underlined) and5′-d(TATTCTAGATCTCTATGACGATTATGAA)-3′ (SEQ ID NO: 4; XbaI restrictionsite underlined), respectively.

CH12 was prepared by ligating a PCR fragment corresponding to the first1326 nucleotides of CH4 with the ClaI/KpnI fragment of the TEAS geneinto the EcoRI/KpnI sites of the pGBT-T19 vector. The CH4 fragment wasprepared using forward amplification primer5′-d(GGGAGCTCGAATTCCATGGCCTCAGCAGCAGTTGCAAACTAT)-3′ (SEQ ID NO: 5; EcoRIrestriction site underlined and translation start codon in bold) andreverse primer 5′-d(GGGATCGATAACTCTGCATAATGTAGCATT)-3′ (SEQ ID NO: 6;ClaI restriction site underlined).

Chimeric synthases CH6, CH8, CH9, CH10, CH11, and CH14 were constructedas follows. Ligation of the EcoRI/HindIII fragment of the HVS gene withthe HindIII/KpnI fragment of CH3 generated CH6. CH8 was created byligating the EcoRI/NdeI fragment of HVS with the NdeI/KpnI fragment ofCH3. CH9 was created by ligating the EcoRI/NdeI fragment of CH5 with theNdeI/KpnI fragment of HVS. CH10 was constructed by ligating theEcoRI/HindIII fragment of HVS with the HindIII/KpnI fragment of CH4.CH11 was constructed by ligating the EcoRI/NdeI fragment of HVS with theNdeI/KpnI fragment of CH4. And CH14 was generated by substituting theEcoRI/Ndel fragment of CH13 with the corresponding DNA fragment ofpBSK-HVS. The nucleotide junctions of the chimeric constructs wereconfirmed by double-stranded DNA sequencing using the dideoxy nucleotidechain termination kit, according to the manufacturer's instructions(U.S. Biochemical Corp).

Chimeric synthases were expressed in E. coli TB1 cells. Procedures forgrowth of the bacterial cells, induction of gene expression, measurementof sesquiterpene synthase enzyme activity, and the determination oftotal protein in the bacterial lysates were performed according to themethods described by Back and Chappell (Arch. Biochem. Biophys. 315:527,1994; J. Biol. Chem. 270:7375, 1995). Reaction products were separatedby developing G60 silica TLC plates impregnated with 15% silver nitratein benzene:hexane:diethyl ether (50:50:1). For qualitative evaluations,TLC plates were sprayed with Enhance surface fluorography spray (Dupont)and exposed to Kodak XAR-5 film for 2 to 5 days at −70° C. Forquantitative evaluations, 0.5 mm zones of an entire lane from a TLCplates were scraped into scintillation vials, and the radioactivity wasdetermined using a Packard 1500 Liquid Scintillation Counter. Thedominant reaction products generated by the synthase activitiesresulting from expression of the TEAS, HVS, CH4, and CH14 constructs inbacterial lysates were also verified by gas chromatography (GC) and gaschromatography-mass spectroscopy (GC-MS) according to the conditionsdescribed by Chappell et al. (Phytochemistry 26:2259, 1987) (data notshown). In addition, mass spectra profiles were compared to thatpublished for 5-epi-aristolochene (Anke and Sterner, Planta Med. 57:344,1991) and the predicted fragmentation pattern for vetispiradiene (Enzellet al., Mass Spectrometry Rev. 3:395, 1984).

As shown in FIGS. 4A-B, the dominant reaction product resulting from theexpression of the tobacco TEAS gene expressed was 5-epi-aristolochene,and vetispiradiene was found to be the dominant reaction productresulting from the expression of the HVS gene. The predominant reactionproducts generated by the expression of CH1 and CH2 were alsoHVS-specific (i.e., vetispiradiene), with enzyme specific activitiessimilar to those found for HVS that was expressed from the pBSK-HVSplasmid. These results indicated that the amino-terminal half of TEASand HVS were functionally equivalent with respect to the HVScarboxy-terminus and do not contribute to the specificity of thereaction product. CH7, having an HVS amino terminus and a TEAS carboxyterminus, is the converse construct of CH2, and the resulting synthaseactivity was expected to result in expression of a TEAS-specific product(i.e., 5-epi-aristolochene). Immunodetection assays revealed thatsynthase protein produced upon expression of CH7 was found to be of thecorrect size and expected abundance (data not shown); however, no enzymeactivity was detected. The lack of enzyme activity indicated thatinteractions between the carboxy and amino terminal portions of theprotein contributed to enzyme activity. This interpretation is furthersupported by comparing the specific activity of the enzymes generated bythe expression of the CH5 and CH6 constructs. CH5 resulted in theexpression of a product having a 10-fold lower specific activity ofsynthase enzyme activity than the other chimeric synthases, even thoughthe absolute level of expressed protein was similar to the otherconstructs (as determined by immunodetection, data not shown).Substituting an HVS carboxy-terminal region was found to restore thespecific activity to the synthase enzyme that was generated by CH6.

Comparison of CH2 and CH3 chimeric synthases provided evidence forspecificity of end-product formation residing within a domain ofapproximately 181 amino acids, corresponding to the NdeI and ClaIrestriction sites within the TEAS and HVS genes. Expression of CH4unexpectedly resulted in the production of a chimeric synthase proteincapable of generating reaction products reflective of both the TEAS andHVS enzymes. We interpreted this result to indicate that amino acids 261to 379 within the tobacco 5-epi-aristolochene synthase are responsiblefor the TEAS-specific products (i.e., the region corresponding to theNdeI to HincII fragment of the cDNA), while amino acids 379 to 442within the Hyoscyamus protein are responsible for the HVS-specificproducts (i.e., the region corresponding to the HincII to ClaI fragmentof the cDNA).

Our interpretation was confirmed by evaluating the expression productsof CH11 and CH12. CH11 represented the substitution of the NdeI toHincII fragment of the Hyoscyamus gene with the corresponding tobaccogene fragment, and resulted in the production of an enzyme havingHVS-and TEAS-specificity. CH12 represented a substitution of the HincIIto ClaI fragment of the tobacco gene with the corresponding Hyoscyamusgene fragment, and resulted in the production of an enzyme havingHVS-and TEAS-specificity. Comparing CH11 to CH13 provided a furtherrefinement in the domain characterization of the tobacco enzymeresponsible for the TEAS-specific products. The fact that CH13 was foundto be a multifunctional enzyme indicated that the 81 amino acids encodedby the DNA fragment residing between the NdeI to XbaI restriction sitesof the tobacco cDNA were sufficient for formation of the predominantTEAS specific products. This interpretation was confirmed bysubstituting the domain contained within the NdeI/XbaI HVS cDNArestriction fragment of CH14 with that of the TEAS gene (FIG. 4A).

As shown in FIG. 4B, the predominant reaction product(s) of the wildtypetobacco TEAS and Hyoscyamus HVS genes expressed in bacteria migrated onsilver nitrate-TLC plates with R_(f) values of 0.41 and 0.31, valuesconsistent with previous characterization of these products as5-epi-aristolochene and vetispiradiene, respectively (Back and Chappell,J Biol. Chem. 270:7375, 1995; Back et al., Arch. Biochem. Biophys.315:527, 1994). GC and GC-MS analyses indicated that the predominantTEAS reaction products were 5-epi-aristolochene (70% of total products,based on percentage of total peak areas from GC analysis) and a bicyclicsesquiterpene (20%) ([M]⁺ion at m/z of 204). The predominant HVSreaction product was vetispiradiene (>90%) ([M]⁺ion at m/z of 204 with abase peak at m/z 41 and a series of predictable ions at m/z 175, 108,94, and 68), and the predominant reaction products of CH4 were5-epi-aristolochene (18%), a bicyclic sesquiterpene (43%), andvetispiradiene (32%) (data not shown).

In addition, studies relying on affinity purification ofhistidine-tagged recombinant synthase proteins has revealed five otherminor reaction products, each representing approximately 1% of the totalproducts, with all five found at the same relative abundance in all thereaction assays.

Ratio-Determinant Domain

Another domain of the synthase proteins was identified by comparing therelative ratio of the predominant reaction products produced by themultifunctional chimeric synthase enzymes (FIG. 4A). For example, thereaction products resulting from expression of constructs CH4, CH10,CH11, and CH12 were generated in a ratio of 60-70% TEAS-specific to30-40% HVS-specific. In contrast, an inverse ratio of reaction productsresulted from expression of constructs CH13 and CH14. This resultindicated that the region encompassed by the XbaI to HincII domaininfluenced the relative ratio of reaction products generated by themultifunctional chimeric synthase enzymes. These results indicated thattwo separate and distinct domains within the synthase peptidecontributed directly to the types of reaction products generated, andare interrupted by another domain which we refer to as theratio-determinant domain (FIG. 5).

Site-Directed Mutagenesis

Additional analysis of the product specificity and ratio determinantdomains was determined using conventional site-directed mutagenesismethodologies. The results of this analysis are presented in Table I(below). For example, the DDXXD motif, found within the aristolochenespecific domain, is a conserved sequence that is found in a variety ofterpene biosynthetic enzymes including TEAS and HVS. This acidic aminoacid cluster is said to coordinate a metal cofactor that is necessary toneutralize the diphosphate moiety of FPP in an otherwise lipophilicpocket. Substitution of the first aspartic acid residue (D301) of theDDXXD motif with either glutamic acid (overall charge conservation) orvaline (net loss of acidic charge) residues (i.e., D301→E and D301→V)resulted in the formation of an inactivated enzyme. A conservedsubstitution of the second aspartic acid (D302) with a glutamic acidresidue (i.e., D302→E) also inactivated chimeric synthase enzymeactivity by 95%, and resulted in a slight alteration of the productdistribution of the multifunctional enzyme.

TABLE I Mutated Specific Mutation amino Product ratio Activity targetgene acid Aristolochene Vetispiradiene (nmol/mg h⁻¹) CH4 66% 34% 34Substrate binding domain (Ndel/Xbal region) Tobacco D301V No activity 0CH4 R287A No activity 0 CH4 D301V No activity 0 CH4 D301E No activity 0CH4 D302E 51% 49% 1.8 Ratio determinant domain (Xbal/HincII region) CH4K347I 64% 36% 32 CH4 H360S 63% 32% 29 CH4 H364S 65% 35% 38 Hyoscyamusspecific domain (HincII/ClaI region) CH4 T408A 67% 33% 48 CH4 K420M 68%32% 29 CH4 H422A 67% 33% 30 CH4 N436S 70% 30% 32 CH4 AT437, 61% 39% 33438VI

The sites for directed substitutions within the ratio-determinant domain(i.e., K347→I, H360→S, H364→S) were inferred by an analysis of reportsthat hypothesized the importance of charged amino acid residues (e.g.,histidine or lysine) in synthase enzymology, and these sites representedthose amino acids which displayed the greatest charge differences incomparisons between the TEAS and HVS primary sequences. None of thethree mutations analyzed had any effect on overall catalytic activity orthe ratio of products formed.

Amino acid substitutions within the HVS specific domain were chosen onthe basis of comparisons between secondary structural predictions of theHVS and TEAS proteins. Those amino acids mutated appeared to contributedisproportionately to structural distortions in the secondary structuremodels of these two proteins, largely because of charge considerations.However, as shown in Table I (above), substitutions involving charged tonon-charged (i.e., T408→A, K420→M, H422→A) or reduced charged (N436→S,A437→T, V438→I) amino acids did not affect overall enzyme activity, northe synthesis rate of one product or the other.

Quiescent Synthases

To generate a quiescent synthase, the inactive domain corresponding toexon 4 of HVS is substituted with the corresponding active domain ofCH3, as outlined in FIG. 6. CH3 contains an inactive domaincorresponding to exon 6 of TEAS, has convenient NdeI and XbaIrestriction sites for the desired substitution, and can be overexpressedin bacteria to high levels. Domain switching is accomplished usingstandard molecular techniques, as described herein. In one particularexample, a PCR amplification product of HVS cDNA corresponding to exon4, encompassing amino acids 261 to 342 and containing appropriate NdeIand XbaI sites within the primers, is substituted for the correspondingregion of CH3. In generating such constructs, care is exercised tomaintain appropriate amino acid residues and the correct reading frame,and expression testing of the construct entails a measurement of theprotein level in the soluble and insoluble fractions of bacteriallysates by immunoblotting techniques, as well as by enzyme assays.

In addition, large scale enzyme reactions are performed, the reactionproduct(s) are extracted into hexane, and the products are purified byHPLC methods. Additional evaluation of the quiescent enzyme reactionproducts is carried out using TLC, and comparing the Rf values of theexperimental sample to those generated by the TEAS and HVS enzymes.Retention times of the reaction products (e.g., germacrene orgermacrene-like reaction product) are also monitored using GC, GC-MS,and NMR according to standard methods.

The quiescent synthase is useful for providing sufficient amounts of thegermacrene reaction intermediate(s) (or derivatives thereof) toconfirming the chemical rationalization for the EAS and VS reactions,and produces a template chimeric synthase that may be used for theintroduction of novel terminal steps in the overall synthase reactionscheme.

Chimeric Casbene and Cadinene Synthases

Chimeric isoprenoid synthases are also useful for generating novelmacrocyclic isoprenoids or isoprenoids having altered stereochemicalproperties. For example, isoprenoid synthases such as casbene synthase,a diterpene synthase which catalyzes the synthesis of a macrocyclicditerpene harboring a cyclopropyl side group, and cadiene synthase, asesquiterpene synthase that catalyzes the synthesis of a bicyclicsesquiterpene, provide domains useful for engineering enzymes capable ofproducing macrocylic isoprenoids or isoprenoid reaction products havingaltered sterochemical properties. A general scheme for producing suchchimeric synthases is presented in FIGS. 7 and 8.

To construct such chimeric casbene and cadienne synthases, quiescentamino terminal domains (and other synthase domains as necessary) aresubstituted with those from casbene and cadinene synthase usingconvenient restriction sites and PCR amplification of selected regionsas described above. Sequences corresponding to the N-terminal, plastidtargeting sequence of the casbene synthase are deleted in theseconstructs. Chimeric constructs are expressed in bacteria, bacteriallysates are examined for chimeric synthase activity, and reactionproducts are characterized as described above, for example, usingargentation-TLC. Constructs supporting high levels of synthase activityin bacteria and/or activity generating reaction products which migratewith Rƒs different from aristolochene and vetispiradiene standards areconsidered useful in the invention. Reaction products are also analyzedfor their retention times by GC and subjected to GC-MS and NMR, asnecessary. Those domains of casbene synthase and cadinene synthase whichcontribute to the synthesis of unique reaction products may also besubjected to fine detail mapping using a strategy analogous to thatdepicted in FIG. 4A.

Production of Other Chimeric Isoprenoid Synthases

Using the standard molecular techniques described herein, other chimericsynthases may be readily generated which include domains from known ornewly isolated synthase enzymes. Such chimeric synthases may be testedfor activity using, for example, any appropriate enzyme assays known tothose in the art, or by standard immunodetection techniques.

The isolation of additional synthase coding sequences is also possibleusing standard cloning strategies and techniques that are well known inthe art. For example, using all or a portion of the amino acid sequenceof a known synthase polypeptide, one may readily designsynthase-specific oligonucleotide probes, including synthase degenerateoligonucleotide probes (i.e., a mixture of all possible coding sequencesfor a given amino acid sequence). These oligonucleotides may be basedupon the sequence of either DNA strand and any appropriate portion ofsynthase nucleotide sequence. General methods for designing andpreparing such probes are provided, for example, in Ausubel et al.,1996, Current Protocols in Molecular Biology, Wiley Interscience, NewYork, and Berger and Kimmel, Guide to Molecular Cloning Techniques,1987, Academic Press, New York. These oligonucleotides are useful forsynthase gene isolation, either through their use as probes capable ofhybridizing to a synthase complementary sequences or as primers forvarious amplification techniques, for example, polymerase chain reaction(PCR) cloning strategies.

Hybridization techniques and screening procedures are well known tothose skilled in the art and are described, for example, in Ausubel etal. (supra); Berger and Kimmel (supra); Chen at al. Arch. Biochem.Biophys. 324:255, 1995; and Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory Press, New York. Ifdesired, a combination of different oligonucleotide probes may be usedfor the screening of a recombinant DNA library. The oligonucleotides maybe detectably-labeled using methods known in the art and used to probefilter replicas from a recombinant DNA library. Recombinant DNAlibraries are prepared according to methods well known in the art, forexample, as described in Ausubel et al. (supra), or they may be obtainedfrom commercial sources.

As discussed above, synthase oligonucleotides may also be used asprimers in amplification cloning strategies, for example, using PCR. PCRmethods are well known in the art and are described, for example, in PCRTechnology, Erlich, ed., Stockton Press, London, 1989; PCR Protocols: AGuide to Methods and Applications, Innis et al., eds., Academic Press,Inc., New York, 1990; and Ausubel et al. (supra). Primers are optionallydesigned to allow cloning of the amplified product into a suitablevector, for example, by including appropriate restriction sites at the5′ and 3′ ends of the amplified fragment (as described herein). Ifdesired, a synthase gene may be isolated using the PCR “RACE” technique,or Rapid Amplification of cDNA Ends (see, e.g., Innis et al. (supra)).By this method, oligonucleotide primers based on a synthase sequence areoriented in the 3′ and 5′ directions and are used to generateoverlapping PCR fragments. These overlapping 3′- and 5′-end RACEproducts are combined to produce an intact full-length cDNA. This methodis described in Innis et al. (supra); and Frohman et al., Proc. Natl.Acad. Sci. USA 85:8998, (1988).

Useful synthase sequences may be isolated from any appropriate organism.Confirmation of a sequence's relatedness to the synthase polypeptidefamily may be accomplished by a variety of conventional methods, forexample, sequence comparison. In addition, the activity of any synthaseprotein may be evaluated according to any of the techniques describedherein.

Chimeric Isoprenoid Synthase Polypeptide Expression

Chimeric synthase polypeptides may be produced by transformation of asuitable host cell with all or part of a chimeric synthase DNA (forexample, the chimeric synthase cDNAs described above) in a suitableexpression vehicle or with a plasmid construct engineered for increasingthe expression of a chimeric synthase polypeptide in vivo.

Those skilled in the field of molecular biology will appreciate that anyof a wide variety of expression systems may be used to provide therecombinant protein. The precise host cell used is not critical to theinvention. The chimeric synthase protein may be produced in aprokaryotic host, for example, E. coli TB1, or in a eukaryotic host, forexample, Saccharomyces cerevisiae, mammalian cells (for example, COS 1or NIH 3T3 cells), or any of a number of plant cells including, withoutlimitation, algae, tree species, ornamental species, temperate fruitspecies, tropical fruit species, vegetable species, legume species,monocots, dicots, or in any plant of commercial or agriculturalsignificance. Particular examples of suitable plant hosts include, butare not limited to, Conifers, Petunia, Tomato, Potato, Tobacco,Arabidopsis, Lettuce, Sunflower, Oilseed rape, Flax, Cotton, Sugarbeet,Celery, Soybean, Alfalfa, Medicago, Lotus, Vigna, Cucumber, Carrot,Eggplant, Cauliflower, Horseradish, Morning Glory, Poplar, Walnut,Apple, Asparagus, Rice, Maize, Millet, Onion, Barley, Orchard grass,Oat, Rye, and Wheat.

Such cells are available from a wide range of sources including: theAmerican Type Culture Collection (Rockland, Md.); or from any of anumber seed companies, for example, W. Atlee Burpee Seed Co.(Warminster, Pa.), Park Seed Co. (Greenwood, S.C.), Johnny Seed Co.(Albion, Me.), or Northrup King Seeds (Harstville, S.C.). Descriptionsand sources of useful host cells are also found in Vasil I.K., CellCulture and Somatic Cell Genetics of Plants, Vol I, II, III LaboratoryProcedures and Their Applications Academic Press, New York, 1984; Dixon,R.A., Plant Cell Culture-A Practical Approach, IRL Press, OxfordUniversity, 1985; Green et al., Plant Tissue and Cell Culture, AcademicPress, New York, 1987; and Gasser and Fraley, Science 244:1293, (1989).

For prokaryotic expression, DNA encoding a chimeric synthase polypeptideis carried on a vector operably linked to control signals capable ofeffecting expression in the prokaryotic host. If desired, the codingsequence may contain, at its 5′ end, a sequence encoding any of theknown signal sequences capable of effecting secretion of the expressedprotein into the periplasmic space of the host cell, therebyfacilitating recovery of the protein and subsequent purification.Prokaryotes most frequently used are various strains of E. coli;however, other microbial strains may also be used. Plasmid vectors areused which contain replication origins, selectable markers, and controlsequences derived from a species compatible with the microbial host.Examples of such vectors are found in Pouwels et al. (supra) or Ausubelet al. (supra). Commonly used prokaryotic control sequences (alsoreferred to as “regulatory elements”) are defined herein to includepromoters for transcription initiation, optionally with an operator,along with ribosome binding site sequences. Promoters commonly used todirect protein expression include the beta-lactamase (penicillinase),the lactose (lac), the tryptophan (Trp) (Goeddel et al., Nucl. AcidsRes. 8:4057 (1980)), and the tac promoter systems, as well as thelambda-derived P_(L) promoter and N-gene ribosome binding site (Simatakeet al., Nature 292:128 (1981)).

One particular bacterial expression system for chimeric synthasepolypeptide production is the E. coli pET expression system (Novagen).According to this expression system, DNA encoding a chimeric synthasepolypeptide is inserted into a pET vector in an orientation designed toallow expression. Since the chimeric synthase gene is under the controlof the T7 regulatory signals, expression of chimeric synthase is inducedby inducing the expression of T7 RNA polymerase in the host cell. Thisis typically achieved using host strains which express T7 RNA polymerasein response to IPTG induction. Once produced, recombinant chimericsynthase polypeptide is then isolated according to standard methodsknown in the art, for example, those described herein.

Another bacterial expression system for chimeric synthase polypeptideproduction is the pGEX expression system (Pharmacia). This systememploys a GST gene fusion system that is designed for high-levelexpression of a gene or gene fragment as a fusion protein with rapidpurification and recovery of the functional gene product. The chimericsynthase protein of interest is fused to the carboxyl terminus of theglutathione S-transferase protein protein from Schistosomajaponicum andis readily purified from bacterial lysates by affinity chromatographyusing Glutathione Sepharose 4B. Fusion proteins can be recovered undermild conditions by elution with glutathione. Cleavage of the glutathioneS-transferase domain from the fusion protein is facilitated by thepresence of recognition sites for site-specific proteases upstream ofthis domain. For example, proteins expressed in pGEX-2T plasmids may becleaved with thrombin; those expressed in pGEX-3X may be cleaved withfactor Xa.

For eukaryotic expression, the method of transformation or transfectionand the choice of vehicle for expression of the chimeric synthasepolypeptide will depend on the host system selected. Transformation andtransfection methods of numerous organisms, for example, the baker'syeast Saccharomyces cerevisiae, are described, e.g., in Ausubel et al.(supra); Weissbach and Weissbach, Methods for Plant Molecular Biology,Academic Press, 1989; Gelvin et al., Plant Molecular Biology Manual,Kluwer Academic Publishers, 1990; Kindle, K., Proc. Natl. Acad. Sci.U.S.A 87:1228 (1990); Potrykus, I., Annu. Rev. Plant Physiol. Plant Mol.Biology 42:205 (1991); and BioRad (Hercules, Calif.) Technical Bulletin#1687 (Biolistic Particle Delivery Systems). Expression vehicles may bechosen from those provided, e.g., in Cloning Vectors: A LaboratoryManual (P. H. Pouwels et al., 1985, Supp. 1987); Gasser and Fraley(supra); Clontech Molecular Biology Catalog (Catalog 1992/93 Tools forthe Molecular Biologist, Palo Alto, Calif.); and the references citedabove.

One preferred eukaryotic expression system is the mouse 3T3 fibroblasthost cell transfected with a pMAMneo expression vector (Clontech).pMAMneo provides: an RSV-LTR enhancer linked to adexamethasone-inducible MMTV-LTR promotor, an SV40 origin of replicationwhich allows replication in mammalian systems, a selectable neomycingene, and SV40 splicing and polyadenylation sites. DNA encoding achimeric synthase polypeptide is inserted into the pMAMneo vector in anorientation designed to allow expression. The recombinant chimericsynthase polypeptide is then isolated as described below. Otherpreferable host cells which may be used in conjunction with the pMAMneoexpression vehicle include COS cells and CHO cells (ATCC Accession Nos.CRL 1650 and CCL 61, respectively).

Alternatively, if desired, a chimeric synthase polypeptide is producedby a stably-transfected mammalian cell line. A number of vectorssuitable for stable transfection of mammalian cells are available to thepublic, e.g., see Pouwels et al. (supra); methods for constructing suchcell lines are also publicly available, e.g., in Ausubel et al. (supra).In one example, cDNA encoding the chimeric synthase polypeptide iscloned into an expression vector which includes the dihydrofolatereductase (DHFR) gene. Integration of the plasmid and, therefore, thechimeric synthase-encoding gene into the host cell chromosome isselected for by inclusion of 0.01-300 μM methotrexate in the cellculture medium (as described in Ausubel et al., supra). This dominantselection can be accomplished in most cell types. Recombinant proteinexpression can be increased by DHFR-mediated amplification of thetransfected gene. Methods for selecting cell lines bearing geneamplifications are described in Ausubel et al. (supra); such methodsgenerally involve extended culture in medium containing graduallyincreasing levels of methotrexate. DHFR-containing expression vectorscommonly used for this purpose include pCVSEII-DHrF and pAdD26SV(A)(described in Ausubel et al., supra). Any of the host cells describedabove or, preferably, a DHFR-deficient CHO cell line (for example, CHODHFR-cells, ATCC Accession No. CRL 9096) are among the host cellspreferred for DHFR selection of a stably-transfected cell line orDHFR-mediated gene amplification.

A chimeric synthase polypeptide is preferably produced by astably-transfected plant cell line or by a transgenic plant. A number ofvectors suitable for stable transfection of plant cells or for theestablishment of transgenic plants are available to the public; suchvectors are described in Pouwels et al. (supra), Weissbach and Weissbach(supra), and Gelvin et al. (supra). Methods for constructing such celllines are described in, e.g., Weissbach and Weissbach (supra), andGelvin et al. (supra). Typically, plant expression vectors include (1) acloned chimeric synthase gene under the transcriptional control of 5′and 3′ regulatory sequences and (2) a dominant selectable marker. Suchplant expression vectors may also contain, if desired, a promoterregulatory region (for example, one conferring inducible or constitutiveexpression, or environmentally- or developmentally-regulated, orpathogen- or wound-inducible, or cell- or tissue-specific expression), atranscription initiation start site, a ribosome binding site, an RNAprocessing signal, a transcription termination site, and/or apolyadenylation signal.

The chimeric synthase DNA sequence of the invention may, if desired, becombined with other DNA sequences in a variety of ways. The chimericsynthase DNA sequence of the invention may be employed with all or partof the gene sequences normally associated with a synthase protein. Inits component parts, a DNA sequence encoding a chimeric synthase proteinis combined in a DNA construct having a transcription initiation controlregion capable of promoting transcription and translation in a hostcell.

In general, the constructs will involve regulatory regions functional inplants which provide for production of a chimeric synthase protein asdiscussed herein. The open reading frame coding for the chimericsynthase protein or functional fragment thereof will be joined at its 5′end to a transcription initiation regulatory region such as the sequencenaturally found in the 5′ upstream region of a synthase structural gene.Numerous other transcription initiation regions are available whichprovide for constitutive or inducible regulation.

For applications when developmental, cell, tissue, hormonal,environmental, or pathogen-inducible expression are desired, appropriate5′ upstream non-coding regions are obtained from other genes; forexample, from genes regulated during seed development, embryodevelopment, leaf development, or in response to a pathogen.

Regulatory transcript termination regions may also be provided in DNAconstructs of this invention as well. Transcript termination regions maybe provided by the DNA sequence encoding a synthase protein or anyconvenient transcription termination region derived from a differentgene source. The transcript termination region will contain preferablyat least 1-3 kb of sequence 3′ to the structural gene from which thetermination region is derived. Such genetically-engineered plants areuseful for a variety of industrial and agricultural applications asdiscussed below. Importantly, this invention is applicable togymnosperms and angiosperms, and will be readily applicable to any newor improved transformation or regeneration method.

An example of a useful plant promoter according to the invention is acaulimovirus promoter, for example, a cauliflower mosaic virus (CaMV)promoter. These promoters confer high levels of expression in most planttissues, and the activity of these promoters is not dependent on virallyencoded proteins. CaMV is a source for both the 35S and 19S promoters.In most tissues of transgenic plants, the CaMV 35S promoter is a strongpromoter (see, e.g., Odell et al., Nature 313:810 (1985)). The CaMVpromoter is also highly active in monocots (see, e.g., Dekeyser et al.,Plant Cell 2:591 (1990); Terada and Shimamoto, Mol. Gen. Genet. 220:389,(1990)). Moreover, activity of this promoter can be further increased(i.e., between 2-10 fold) by duplication of the CaMV 35S promoter (seee.g., Kay et al., Science 236:1299 (1987); Ow et al., Proc. Natl. Acad.Sci. U.S.A. 84:4870 (1987); and Fang et al., Plant Cell 1:141 (1989)).

Other useful plant promoters include, without limitation, the nopalinesynthase promoter (An et al., Plant Physiol. 88:547 (1988)) and theoctopine synthase promoter (Fromm et al., Plant Cell 1:977 (1989)).

For certain applications, it may be desirable to produce the chimericsynthase gene product in an appropriate tissue, at an appropriate level,or at an appropriate developmental time. For this purpose, there are anassortment of gene promoters, each with its own distinct characteristicsembodied in its regulatory sequences, shown to be regulated in responseto the environment, hormones, and/or developmental cues. These includegene promoters that are responsible for heat-regulated gene expression(see, e.g., Callis et al., Plant Physiol. 88:965 (1988); Takahashi andKomeda, Mol. Gen. Genet. 219:365 (1989); and Takahashi et al. Plant J.2:751 (1992)), light-regulated gene expression (e.g., the pea rbcS-3Adescribed by Kuhlemeier et al. (Plant Cell 1:471 (1989); the maize rbcSpromoter described by Schaffner and Sheen, (Plant Cell 3:997(1991); orthe chlorophyll a/b-binding protein gene found in pea described bySimpson et al. (EMBO J 4:2723 (1985)), hormone-regulated gene expression(for example, the abscisic acid (ABA) responsive sequences from the Emgene of wheat described by Marcotte et al. (Plant Cell 1:969 (1989); theABA-inducible HVA1 and HVA22, and the rd29A promoters described forbarley and Arabidopsis by Straub et al. (Plant Cell 6:617 (1994), Shenet al. (Plant Cell 7:295 (1994)), and wound-induced gene expression (forexample, of wunI described by Siebertz et al. (Plant Cell 1:961 (1989)),or organ-specific gene expression (for example, of the tuber-specificstorage protein gene described by Roshal et al. (EMBO J. 6:1155 (1987);the 23-kDa zein gene from maize described by Schernthaner et al. (EMBO J7:1249 (1988); or the French bean 13-phaseolin gene described by Bustoset al. (Plant Cell 1:839 (1989)); and pathogen-inducible gene expressiondescribed by Chappell et al. in U.S. Ser. Nos. 08/471,983, 08/443,639,and 08/577,483, hereby incorporated by reference.

Plant expression vectors may also optionally include RNA processingsignals, for example, introns, which have been shown to be important forefficient RNA synthesis and accumulation (Callis et al., Genes and Dev.1:1183 (1987)). The location of the RNA splice sequences candramatically influence the level of transgene expression in plants. Inview of this fact, an intron may be positioned upstream or downstream ofa chimeric synthase polypeptide-encoding sequence in the transgene tomodulate levels of gene expression.

In addition to the aforementioned 5′ regulatory control sequences, theexpression vectors may also include regulatory control regions which aregenerally present in the 3′ regions of plant genes (Thornburg et al.,Proc. Natl. Acad. Sci. U.S.A. 84:744 (1987); An et al., Plant Cell 1:115(1989)). For example, the 3′ terminator region may be included in theexpression vector to increase stability of the mRNA. One such terminatorregion may be derived from the PI-II terminator region of potato. Inaddition, other commonly used terminators are derived from the octopineor nopaline synthase signals.

The plant expression vector also typically contains a dominantselectable marker gene used to identify those cells that have becometransformed. Useful selectable genes for plant systems include genesencoding antibiotic resistance genes, for example, those encodingresistance to hygromycin, kanamycin, bleomycin, G418, streptomycin, orspectinomycin. Genes required for photosynthesis may also be used asselectable markers in photosynthetic-deficient strains. Alternatively,the green-fluorescent protein from the jellyfish Aequorea victoria maybe used as a selectable marker (Sheen et al., Plant J. 8:777, 1995; Chiuet al., Current Biology 6:325 (1996)). Finally, genes encoding herbicideresistance may be used as selectable markers; useful herbicideresistance genes include the bar gene encoding the enzymephosphinothricin acetyltransferase and conferring resistance to thebroad spectrum herbicide Basta® (Hoechst AG, Frankfurt, Germany).

Efficient use of selectable markers is facilitated by a determination ofthe susceptibility of a plant cell to a particular selectable agent anda determination of the concentration of this agent which effectivelykills most, if not all, of the transformed cells. Some usefulconcentrations of antibiotics for tobacco transformation include, e.g.,75-100 μg/ml (kanamycin), 20-50 μg/ml (hygromycin), or 5-10 μg/ml(bleomycin). A useful strategy for selection of transformants forherbicide resistance is described, e.g., by Vasil et al., supra.

It should be readily apparent to one skilled in the art of molecularbiology, especially in the field of plant molecular biology, that thelevel of gene expression is dependent, not only on the combination ofpromoters, RNA processing signals, and terminator elements, but also onhow these elements are used to increase the levels of selectable markergene expression.

Plant Transformation

Upon construction of the plant expression vector, several standardmethods are available for introduction of the vector into a plant host,thereby generating a transgenic plant. These methods include (1)Agrobacterium-mediated transformation (A. tumefaciens or A. rhizogenes)(see, e.g., Lichtenstein and Fuller In: Genetic Engineering, vol 6, PWJRigby, ed, London, Academic Press, 1987; and Lichtenstein, C.P., andDraper, J, . In: DNA Cloning, Vol II, D. M. Glover, ed, Oxford, IRIPress, 1985)), (2) the particle delivery system (see, e.g., Gordon-Kammet al., Plant Cell 2:603 (1990); or BioRad Technical Bulletin 1687,supra), (3) microinjection protocols (see, e.g., Green et al., supra),(4) polyethylene glycol (PEG) procedures (see, e.g., Draper et al.,Plant Cell Physiol. 23:451 (1982); or e.g., Zhang and Wu, Theor. Appl.Genet. 76:835 (1988)), (5) liposome-mediated DNA uptake (see, e.g.,Freeman et al., Plant Cell Physiol. 25:1353 (1984)), (6) electroporationprotocols (see, e.g., Gelvin et al., supra; Dekeyser et al., supra;Fromm et al., Nature 319:791 (1986); Sheen, Plant Cell 2:1027 (1990); orJang and Sheen Plant Cell 6:1665 (1994)), and (7) the vortexing method(see, e.g., Kindle supra). The method of transformation is not criticalto the present invention. Any method which provides for efficienttransformation may be employed. As newer methods are available totransform crops or other host cells, they may be directly applied.

The following is an example outlining one particular technique, anAgrobacterium-mediated plant transformation. By this technique, thegeneral process for manipulating genes to be transferred into the genomeof plant cells is carried out in two phases. First, cloning and DNAmodification steps are carried out in E. coli, and the plasmidcontaining the gene construct of interest is transferred by conjugationor electroporation into Agrobacterium. Second, the resultingAgrobacterium strain is used to transform plant cells. Thus, for thegeneralized plant expression vector, the plasmid contains an origin ofreplication that allows it to replicate in Agrobacterium and a high copynumber origin of replication functional in E. coli. This permits facileproduction and testing of transgenes in E. coli prior to transfer toAgrobacterium for subsequent introduction into plants. Resistance genescan be carried on the vector, one for selection in bacteria, forexample, streptomycin, and another that will function in plants, forexample, a gene encoding kanamycin resistance or herbicide resistance.Also present on the vector are restriction endonuclease sites for theaddition of one or more transgenes and directional T-DNA bordersequences which, when recognized by the transfer functions ofAgrobacterium, delimit the DNA region that will be transferred to theplant.

In another example, plant cells may be transformed by shooting into thecell tungsten microprojectiles on which cloned DNA is precipitated. Inthe Biolistic Apparatus (Bio-Rad) used for the shooting, a gunpowdercharge (22 caliber Power Piston Tool Charge) or an air-driven blastdrives a plastic macroprojectile through a gun barrel. An aliquot of asuspension of tungsten particles on which DNA has been precipitated isplaced on the front of the plastic macroprojectile. The latter is firedat an acrylic stopping plate that has a hole through it that is toosmall for the macroprojectile to pass through. As a result, the plasticmacroprojectile smashes against the stopping plate, and the tungstenmicroprojectiles continue toward their target through the hole in theplate. For the present invention, the target can be any plant cell,tissue, seed, or embryo. The DNA introduced into the cell on themicroprojectiles becomes integrated into either the nucleus or thechloroplast.

In general, transfer and expression of transgenes in plant cells are nowroutine practices to those skilled in the art, and have become majortools to carry out gene expression studies in plants and to produceimproved plant varieties of agricultural or commercial interest.

Transgenic Plant Regeneration

Plants cells transformed with plant expression vectors can beregenerated, for example, from single cells, callus tissue, or leafdiscs according to standard plant tissue culture techniques. It is wellknown in the art that various cells, tissues, and organs from almost anyplant can be successfully cultured to regenerate an entire plant; suchtechniques are described, e.g., in Vasil supra; Green et al., supra;Weissbach and Weissbach, supra; and Gelvin et al., supra.

In one particular example, a cloned chimeric synthase polypeptide underthe control of the EAS4 promoter and the nopaline synthase terminatorand carrying a selectable marker (for example, kanamycin resistance) istransformed into Agrobacterium. Transformation of leaf discs (forexample, of tobacco leaf discs), with vector-containing Agrobacterium iscarried out as described by Horsch et al. (Science 227:1229 (1985)).Putative transformants are selected after a few weeks (for example, 3 to5 weeks) on plant tissue culture media containing kanamycin (e.g., 100μg/ml). Kanamycin-resistant shoots are then placed on plant tissueculture media without hormones for root initiation. Kanamycin-resistantplants are then selected for greenhouse growth. If desired, seeds fromself-fertilized transgenic plants can then be sowed in soil-less mediumand grown in a greenhouse. Kanamycin-resistant progeny are selected bysowing surfaced sterilized seeds on hormone-free kanamycin-containingmedia. Analysis for the integration of the transgene is accomplished bystandard techniques (see, for example, Ausubel et al. supra; Gelvin etal. supra).

Transgenic plants expressing the selectable marker are then screened fortransmission of the transgene DNA by standard immunoblot and DNAdetection techniques. Each positive transgenic plant and its transgenicprogeny are unique in comparison to other transgenic plants establishedwith the same transgene. Integration of the transgene DNA into the plantgenomic DNA is in most cases random, and the site of integration canprofoundly effect the levels and the tissue and developmental patternsof transgene expression. Consequently, a number of transgenic lines areusually screened for each transgene to identify and select plants withthe most appropriate expression profiles.

Transgenic lines are generally evaluated for levels of transgeneexpression. Expression at the RNA level is determined initially toidentify and quantitate expression-positive plants. Standard techniquesfor RNA analysis are employed and include PCR amplification assays usingoligonucleotide primers designed to amplify only transgene RNA templatesand solution hybridization assays using transgene-specific probes (see,e.g., Ausubel et al., supra). The RNA-positive plants are then analyzedfor protein expression by Western immunoblot analysis using specificantibodies to the chimeric synthase (see, e.g., Ausubel et al., supra).In addition, in situ hybridization and immunocytochemistry according tostandard protocols can be done using transgene-specific nucleotideprobes and antibodies, respectively, to localize sites of expressionwithin transgenic tissue.

Once the recombinant chimeric synthase protein is expressed in any cellor in a transgenic plant (for example, as described above), it may beisolated, e.g., using affinity chromatography. In one example, ananti-chimeric synthase antibody (e.g., produced as described in Ausubelet al., supra, or by any standard technique) may be attached to a columnand used to isolate the polypeptide. Lysis and fractionation of chimericsynthase-producing cells prior to affinity chromatography may beperformed by standard methods (see, e.g., Ausubel et al., supra). Onceisolated, the recombinant protein can, if desired, be further purified,for example, by high performance liquid chromatography (see, e.g.,Fisher, Laboratory Techniques In Biochemistry And Molecular Biology,eds., Work and Burdon, Elsevier, 1980).

These general techniques of polypeptide expression and purification canalso be used to produce and isolate useful chimeric synthase fragmentsor analogs.

Use

The invention described herein is useful for a variety of agricultural,pharmaceutical, industrial, and commercial purposes. For example, themethods, DNA constructs, proteins, and transgenic organisms, includingthe bacteria, yeast, and plants described herein, are useful forimproving isoprenoid synthesis, manufacturing, and production.

Our results presented above demonstrate that it is possible to modulateisoprenoid synthase activity by providing chimeric synthases. In thismanner, various synthase reaction products may be modified, controlled,or manipulated, resulting in enhancement of production of numeroussynthase reaction products, for example, the production of novelmonoterpenes, diterpenes, and sesquiterpenes. Such compounds are usefulas phytoalexins, insecticides, perfumes, and pharmaceuticals such asanti-bacterial and fungal agents.

A number of chimeric isoprenoid synthases may be engineered that areuseful, for example, for the production of compounds having anti-fungal,anti-bacterial, anti-malarial, and anti-tumor properties. For example,for the production of chimeric synthases capable of catalyzing theproduction of anti-fungal isoprenoids, the C-terminal domain of casbenesynthase (Mau and West, Proc. Natl. Acad. Sci. 91:8497, 1994) is joinedto the N-terminal domain of TEAS, HVS, or CH9. To produce a chimericsynthase capable of catalyzing the production of anti-bacterialcompounds, the C-terminal domain of cyclofamesenone synthase(Habtermariam et al., J. Nat. Prod. 56:140, 1993) is joined to theN-terminal domain of TEAS, HVS, or CH9. Production of anti-malarialcompounds is achieved using chimeric synthases having a C-terminaldomain from artemisian synthase (El-Feraly et al., J. Nat. Prod. 52:196,1989) and an N-terminal domain from TEAS, HVS, or CH9. Synthases capableof producing anti-tumor compounds are produced by joining the C-terminaldomain of taxadiene synthase (Koepp et al., J. Biol. Chem. 270:8686,1995) or helenalin synthase (Lee et al., Science 196: 533, 1977) withthe N-terminal domain of TEAS, HVS, or CH9.

The invention is also useful for the production of chimeric synthaseswhich are capable of generating insecticides. Such chimeric synthasesare engineered by joining the C-terminal domain of cadenine synthase(Chen et al., Arch. Biochem. Biophys. 324: 255, 1995) to the N-terminaldomain of TEAS, HVS, and CH9.

Finally, chimeric synthases are also useful for generating novelflavorings and perfumes. In one particular example, for the productionof novel flavorings and aromas, a chimeric synthase is engineered byjoining the C-terminal domain of limonene synthase (Colby et al., JBiol. Chem. 268: 23016, 1993) to the C-terminal domain of TEAS, HVS, orCH9.

All publications and patents mentioned in this specification are hereinincorporated by reference to the same extent as if each individualpublication or patent was specifically and individually indicated to beincorporated by reference.

Other Embodiments

From the foregoing description, one skilled in the art can easilyascertain the essential characteristics of this invention, and can makevarious changes and modifications of the invention to adapt it tovarious usages and conditions. Thus, other embodiments are also withinthe claims.

1. DNA encoding a chimeric isoprenoid sesquiterpene synthasepolypeptide, that comprises: a first isoprenoid sesquiterpene synthasepolypeptide domain joined to a second different isoprenoid sesquiterpenesynthase polypeptide domain, interrupted by or including a ratiodeterminant domain, whereby the chimeric isoprenoid sesquiterpenesynthase polypeptide encoded by the DNA catalyzes the production of atleast one isoprenoid sesquiterpene synthase reaction product that is notproduced in the absence of the second isoprenoid sesquiterpene synthasepolypeptide and another isoprenoid sesquiterpene synthase reactionproduct that is produced in the absence of the second sesquiterpenesynthase polypeptide domain.
 2. A vector, comprising the DNA of claim 1.3. A cell, comprising the DNA of claim
 1. 4. The cell of claim 3,wherein said cell is E. coli.
 5. The cell of claim 3, wherein said cellis a plant cell.
 6. A transgenic plant, comprising the DNA of claim 1.7. A vector, comprising the chimeric isoprenoid synthase gene of claim 1and a dominant selectable marker.
 8. The vector of claim 7 furthercomprising additional elements selected from the group consisting of apromoter regulatory region, a transcription initiation start site, aribosome binding site, an RNA processing signal, a transcriptiontermination site, and a polyadenylation agent.
 9. The vector of claim 2comprising a transcription initiation regulatory region.
 10. The vectorof claim 2 comprising a transcription termination regulatory region. 11.The DNA of claim 1, wherein the chimeric isoprenoid sesquiterpenesynthase polypeptide encoded by the DNA includes therein a DDXXD motif.12. A vector comprising the DNA of claim 11.